Semaphorins signaling serves as a path-finding control for the development and homeostasis of the nervous, immune, and cardiovascular systems. The pleiotropic functions of Semaphorins are mediated by their cell surface receptors Plexins, and in many cases aided by the co-receptors Neuropilins. Semaphorins and their receptors are large families of glycoproteins whose respective structures and functions are conserved across the animal kingdom. Dysregulation of Semaphorin signaling has been associated with many pathological conditions, including tumor angiogenesis and progression, neurodegenerative diseases, and multiple sclerosis. The modulation of Semaphorin signaling has therapeutic potential, but is limited by the lack of structural information on Semaphorin-receptor recognition and the activation of Semaphorin receptors. We propose three specific aims to elucidate the molecular mechanisms in Semaphorin signaling: 1) Delineate the basis of class-specific Semaphorin- Plexin recognition; 2) Elucidate the role of Neuropilins in the assembly of class 3 Semaphorin signaling complexes; 3) Understand the mode of Plexin auto-inhibition and the Plexin conformational change induced by Semaphorin binding. Recombinant protein expression and engineering, X-ray crystallography, isothermal titration calorimetry (ITC), and biophysical and cellular assays will be used to accomplish these specific aims. The structural information about Semaphorins and their receptors can serve as a basis for designing potential applications such as anti-angiogenesis and anti- metastasis in cancer treatments, and directional nerve growth at the injury sites. Our research team is highly experienced and has a track record in projects similar to this proposed study. The structural information from this study will be highly novel, and likely will lay the groundwork for novel biochemical and cell biological research in the Semaphorin field.